a) Field of the Invention
This invention is related to immobilization of ligands onto solid surfaces and their use in hybridization, purification, immunoassays, biosensors, and other biochemical applications.
b) Description of Related Art
Solid supports for the immobilization of ligands, such as nucleotides, proteins, enzymes, and cells, are extensively used in hybridization, purification, immunoassays, and many other biochemical applications.
U.S. Pat. No. 5,622,826, issued Apr. 22, 1997, discloses a method by which amino-labeled oligonucleotides are immobilized onto glass by using an isocyanate linker, particularly 1,3-phenylene diisocyanate. This approach suffers from the limitation that 1,3-phenylene diisocyanate is reactive to both hydroxyl and thiol groups, thus lowering dramatically the specificity of the molecule. Further, 1,3-phenylene diisocyanate is a small, inflexible molecule which binds the ligand close to the surface.
Cohen et al. (Nucleic Acids Res., 1997, 25(4), 911–912) disclose a method for immobilizing oligonucleotides to glass using phosphite-triester chemistry for solid phase oligonucleotide synthesis. The phosphite-triester molecules bind multiple hydroxyl groups on the glass surface and the phosphate group at the 5′-end of the nucleotide. Although this approach provides a stable covalent bond to the surface, it has the limitations of binding the ligand close to the surface, thus lowering the exposure of the ligand, as well as occupying three hydroxyl groups per ligand, thus lowering the surface density of ligand.
Alkylsiloxanes are one of the most widely used classes of molecules for activating glass surfaces with functional groups (Weetall, H. H., Appl. Biochem. Biotechnol., 1993, 41, 157–188). These molecules form self-assembled monolayers (SAMs) when the reactive siloxane group condenses with hydroxyl groups of the surface and neighboring siloxanes to form a crosslinked network (Mrksich, M., and Whitesides, G. M., Annu. Rev. Biophys. Biomol. Struct., 1996, 25, 55–78).
In U.S. Pat. No. 5, 837,860, issued Nov. 17, 1998, Anderson and Rogers disclose a method of immobilize single nucleic acids or oligonucleotides labeled with terminal sulfhydryl or disulfide functional groups. Mercaptosilane molecules are first immobilized onto a glass or polystyrene solid surface to which the labeled nucleotides form a covalent disulfide bond, using mercaptoethanol or dithiothreitol as reducing agents.
In U.S. Pat. No. 5,760,130, issued Jun. 2, 1998, Johnston and Trounstine disclose a method for immobilizing DNA using aminoalkylsilanes. After the aminoalkylsilanes are immobilized on the glass surface, a carbodiimide solution in an imidazole buffer forms an intermediate that reacts with the phosphate group at the 5′-end of DNA. Lom, B., et al., J. Neurosci. Meth., 1993, 50, 385–397 used alkylsiloxanes with a mixture of amino and alkane functionalities to bind proteins by interacting with their hydrophilic and hydrophobic moieties. Others have used alkylsiloxanes functionalized with iodine, benzyl chloride, and epoxide to interact with amino and thiol groups of antibodies (Pope, N. M., et al., Bioconj. Chem., 1993, 4(2), 166–171). Maskos and Southern (Nucleic Acids Res., 1992, 20(7), 1679–1684) used epoxy alkylsilanes and ethylene glycol derivatives to immobilize nucleotides for solid phase synthesis. The epoxy alkylsilanes serve as spacers, while the ethylene glycol derivatives provide hydroxyl groups that are oxidized to react with the phosphate group at the 5′-end of the nucleotide.
Aminoalkylsiloxanes have also been used to immobilize DNA lengthwise on glass surfaces (Yokota et al., Nucleic Acids Res., 1997, 25(5), 1064–1070). The mechanism by which the aminated surface binds DNA is not clear, but is thought to be based on electrostatic interactions. This interaction is far from specific since these aminated surfaces are able to bind any nucleotide sequence. Also, the strength of the interaction is weak, since, after binding, the DNA is straightened by spreading the liquid on the glass surface.
One problem with the use of alkylsiloxanes is that they do not necessarily form SAMs as originally thought (Vandenberg, et al., J. Colloid Inter. Sci., 1991, 147(1), 103–118). Instead of an ordered well-defined structure, they may form aggregates on the surface, thus lowering the surface binding capacity. The structure which alkylsiloxanes form on the glass surface is highly dependent on the reaction conditions.
Another approach for binding DNA lengthwise (or at least at various points across its length) on a glass surface uses poly-1-lysine (Schena, et al., Science, 1995, 270, 467–470, and Shalon, et al., Genome Res., 1996, 6, 639–645). As with the use of aminoalkylsiloxanes, this interaction is not specific and thus weak, resulting in loss of ligand if stringent washing steps are needed.
The interaction of metal ions with specific amino acids on the surface of proteins was first used by Porath et al. (Nature, 1975, 258, 598–607) in chromatography to separate serum proteins using metal ions immobilized by imidoacetate. Following this, most binding studies using metal ions have relied on transition metal ions (e.g., Cu(II), Ni(II), Fe(III), and Zn(II)) which interact with indole and imidazole groups present in proteins.
In U.S. Pat. No. 5,620,850, issued on Apr. 15, 1997, Bamdad et al. attached a construct of a long chain hydroxyalkylthiol and a Ni(II) chelator to a gold surface. Ni(II) is a transition metal ion, which interacts with functional groups present in proteins.
The work by Garcia and co-workers has demonstrated that the soft metal acids Ag(I) and Pt(II) can be used to immobilize proteins and oligonucleotides. Immobilized silver ions have been demonstrated to provide a unique affinity series in the chromatographic separation of amino acids (García, A. A., et al., Reactive Polymers, 1994, 23, 249–259) and a preference of biotin labeled BSA over its unlabeled counterpart (García, A. A., et al., Ind. Eng. Chem. Res., 1996, 35(4), 1097–1106). Also, a biotin labeled nucleotide (b-dUTP) was shown to be retained through affinity interactions, while dUTP was not retained on an immobilized silver ion column when the sodium chloride concentration exceeded 0.001 M (Agarwal, et al., Sep. Sci. Technol., 1998, 33(1), 1–18). Silver ions have also been immobilized onto colloidal paramagnetic particles in order to recover biotin-labeled oligonucleotides from a mixed population (Ramírez-Vick, J. E., and García, A. A., Reactive and Functional Polymers, 1998, 35,123–132).
The use of soft metal ions as anchor groups has been demonstrated when the protein clathrin was immobilized onto a gold surface by using NHS ester-activated dodecanethiols (Wagner, et al., FEBS Letters, 1994, 356, 267–271).
In U.S. Pat. No. 5,622,826, issued on Apr. 22, 1997, Vanna discloses a method for using platinum wafers as a solid surface for immobilizing amino-labeled oligonucleotides, using 1,4-phenylene diisothiocyanate. This molecule lacks the flexibility necessary to be able to bind the labeled ligand at a high surface density while providing the necessary availability to bind the maximum amount of receptor biomolecule possible.
The object of the present invention is to provide an improved method for the immobilization of labeled ligands onto solid surfaces. Several longstanding problems in hybridization, purification, immunoassays, biosensors, and other biochemical applications are resolved by this invention.